AB0026 mtDNA OXIDATIVE DAMAGE IS PROINTERFEROGENIC AND PRONETOTIC, AND RELATES TO ANTIPHOSPHOLIPID ANTIBODY POSITIVITY IN SLE
Authors: S. Giaglis, D. Kyburz, N. Venhoff, U. Walker
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Mitochondria are essential eucaryotic cell organelles with several bacterial features such as a circular genome with hypomethylated CpG areas, and a double cardiolipin membrane [1]. A fundamental role of mitochondria in systemic lupus erythematosus (SLE) was recently shown; mitochondrial ROS participate in neutrophil extracellular traps (NETs) formation [2], while extrusion of cell-free mitochondria and highly oxidised interferogenic mitochondrial DNA (ox-mtDNA) causes disease in an SLE animal model [3, 4]. Moreover, plasma mtDNA is a sensitive biomarker for SLE diagnosis and the clinical monitoring of SLE activity [5]. <h3>Objectives</h3> The present study aimed to explore the cellular mechanisms controlling the inflammatory responses triggered by mtDNA in SLE. <h3>Methods</h3> Total DNA was isolated from healthy individuals’ (HC) and SLE patients’ plasma. Copy numbers were analysed by qPCR for mtDNA (ATP-6) and nuclear DNA (GAPDH). mtDNA was isolated from HC and SLE patients’ platelets. Neutrophils were isolated from EDTA blood by negative selection; cells were incubated with SLE patients’ plasma, and NET formation was assessed by SytoxGreen staining. Tofacitinib, a JAK1-JAK3 inhibitor, was utilised to assess the JAK-STAT pathway activation. DNA oxidation was assessed by MitoSOX Red staining <i>in vitro</i> and 8-hydroxy-diguanosine (8-OHdG) ELISA of plasma. Plasma IFN type 1 was measured by ELISA. Anti-phospholipid antibodies (aPL Abs) comprising of anti-cardiolipin (aCL) and anti-β2GPI, were analysed by ELISA and the lupus anticoagulant (LAC) test was performed. <h3>Results</h3> Neutrophils from healthy donors exhibit enhanced NET formation when incubated with from SLE patients’ plasma (P=0.001), compared to HC plasma. Their NETs are decorated with ox-mtDNA (P=0.01). SLE patients’ mtDNA elicits increased NETs (P=0.001) decorated with ox-mtDNA (P=0.001), which are both not entirely abolished after DNase1 treatment. Formation of NETs and ox-mtDNA extrusion seems to be partially regulated through the type I IFN pathway; JAK inhibition with tofacitinib diminishes, however not entirely, NET formation and ox-mtDNA release (56% and 60% decrease, respectively). mtDNA amounts correlate with 8-OHdG (R<sup>2</sup>=0.87; P&lt;0.0001) and type I IFN levels (R<sup>2</sup>=0.60; P=0.001) in SLE patients’ plasma. aPL Ab-positive SLE patients were characterised by higher plasma levels of mtDNA (P=0.004), but not nDNA, compared to aPL Ab-negative SLE patients. Interestingly, aPL Ab-positive and aCL Ab-positive SLE patients’ plasma demonstrated a higher proNETotic capacity. <h3>Conclusion</h3> Oxidative damage of mtDNA promotes the formation of NETs, is particularly interferogenic, and NETs relate to the antiphospholipid antibody positivity in SLE patients’ circulation. Circulatory ox-mtDNA might promote endothelial damage and vasculitis in SLE. A significant link between aPL, namely aCL IgG, and circulating mtDNA in SLE patients was evident, potentially aggravating the inflammatory state linked to disease severity and promoting thrombotic events. <h3>References</h3> [1]Wang C, Youle RJ. Annu Rev Genet. 2009;43:95-118. [2]West AP, Shadel GS. Nat Rev Immunol. 2017;17:363-75. [3]Crow MK, Olferiev M, Kirou KA. Annu Rev Pathol. 2019;14:369-93. [4]Lood C, Blanco LP, Purmalek MM, Carmona-Rivera C, De Ravin SS, Smith CK, et al. Nat Med. 2016;22:146-53. [5]Giaglis S, Daoudlarian D, Voll RE, Kyburz D, Venhoff N, Walker UA. RMD Open. 2021;7. <h3>Disclosure of Interests</h3> UW is coinventor of patents owned by Freiburg University; NV is coinventor of patents owned by Freiburg University.